Project Summary One of the cardinal features of inflammatory states is that normally innocuous stimuli produce pain. Current pain-relieving drugs include nonsteroidal anti-inflammatory drugs, which are aimed at the interdiction of prostaglandin production, corticosteroids and opioids. However, the side effects and some cases addiction potential associated with these drugs limit their long-term use especially during chronic inflammatory pain. To develop novel, non-addictive analgesics, there remains an urgent need to understand how inflammation produces the change in nociceptor firing that underlies pain perception. In this proposal, we aim to provide proof of principal that in dorsal root ganglion (DRG) neurons, adaptin 2 clathrin-mediated endocytosis (AP2- CME) is a principal facilitator of inflammatory-induced nociceptor sensitization. We have previously demonstrated that in response to protein kinase A (PKA) stimulation, Slack KNa channels are internalized via AP2-CME from DRG neuronal membranes and this caused hyperexcitability. Furthermore we showed that inhibiting AP2-CME prevented PKA-induced neuronal hyperexcitability. Preliminary studies now indicate that in vivo knockdown of the AP2 alpha subunit AP2A2 specifically within DRG neurons, substantially reduces inflammatory pain behavior. Here, we will apply a combination of protein chemistry, immunohistochemistry, electrophysiology, spinal cord physiology, pain behavior assays and a novel in vivo gene knockdown approach to test the hypothesis that AP2-CME is a key regulator of nociceptor sensitization. The specific aims are 1) to determine whether AP2-CME controls basal excitability and neurotransmission 2) To demonstrate that reducing AP2-CME mitigates inflammatory pain. This research project will reveal the central role AP2-CME plays in pain signaling.